EP0103190A2 - Procédé et dispositif de préparation d'une lamelle de microscope portant un échantillon - Google Patents

Procédé et dispositif de préparation d'une lamelle de microscope portant un échantillon Download PDF

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Publication number
EP0103190A2
EP0103190A2 EP83108028A EP83108028A EP0103190A2 EP 0103190 A2 EP0103190 A2 EP 0103190A2 EP 83108028 A EP83108028 A EP 83108028A EP 83108028 A EP83108028 A EP 83108028A EP 0103190 A2 EP0103190 A2 EP 0103190A2
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EP
European Patent Office
Prior art keywords
slide
rotation
axis
support member
sample
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP83108028A
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German (de)
English (en)
Other versions
EP0103190A3 (fr
Inventor
Hasan Ismail Zeya
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Immunomed Corp
Original Assignee
Immunomed Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Immunomed Corp filed Critical Immunomed Corp
Publication of EP0103190A2 publication Critical patent/EP0103190A2/fr
Publication of EP0103190A3 publication Critical patent/EP0103190A3/fr
Withdrawn legal-status Critical Current

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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00Sampling; Preparing specimens for investigation
    • G01N1/28Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/2813Producing thin layers of samples on a substrate, e.g. smearing, spinning-on
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00Sampling; Preparing specimens for investigation
    • G01N1/28Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/2813Producing thin layers of samples on a substrate, e.g. smearing, spinning-on
    • G01N2001/2846Cytocentrifuge method

Definitions

  • the present invention relates to a method and apparatus for preparing samples of liquids for microscopic examination and diagnostic evaluation. More particularly, this invention is concerned with an apparatus and method in which centrifugal force is applied to spread a cell-containing biological liquid sample in a thin streak of single cell thickness on an inclined slide, creating a high quality, undistorted cell smear, having a high numerical density of cells available for differential counting and morphological, histochemical, fluorescent, autoradiographic and various other types of biological tests.
  • the sample on a specimen slide prepared using the wedge technique is thick.
  • the liquid contains cells, this hampers accurate evaluation of the morphology of the cells, which often is a critical factor in the diagnosis of diseases.
  • diagnosis of leukemia it is critical to determine the number, exact type and derivation of leucocytes in the blood. Thick smears of the blood sample on a slide for microscopic examination can cause distortion in the characteristic shape of these cells, causing one cell type to be mistaken for the other. This results in false negative and false positive diagnoses of leukemia.
  • the cells in the samples produced by the method are very few in number and very widely dispersed over almost the entire surface of the slide.
  • Such a wide field for observation also creates inaccuracy and delays in performing differential counts of cell types, since the observer must constantly change the microscopic field in all directions to examine the cells on the entire slide.
  • such wide dispersal also makes it almost impossible to count and identify cells, i.e., blood cells, bacterial cells, or virus particles, in patients with very low numbers of such cells. Because this procedure is slow and tedious, and because it is easy to mistakenly recount, or omit to count, a field many laboratory workers will only count a few fields rather than the entire slide. Thus, the relative number of cell types per amount of sample is frequently inaccurate, another contributing factor to misdiagnosis of the disease in question.
  • a further disadvantage is the relatively large amount of specimen sample required to create the specimen slide. This disadvantage is apparent in cases where the liquid in question is a biological body fluid such as blood, spinal fluid, or serum, and when the patient from whom the specimen is to be taken is either very young or very old and frail.
  • a method of producing a streak of a liquid on a microscope slide, cover slip or the like is intended to refer to the ordinary rectangular glass microscope slide normally denoted by that term, and also other shapes and items, such as square or rectangular cover slips, which can be used to perform the same function.
  • the method of the present invention comprises placing a liquid sample on one side of a slide; positioning the slide at an angle of about 10° to 70° from an axis of rotation with the face of the slide containing the liquid sample facing the axis of rotation, and with the radial distance from the sample to the axis of rotation being less than the greatest radial distance from one end of the slide to the axis of rotation; and spinning the slide around the axis of rotation so that it travels in a path which defines a truncated conical shell, whereby the centrifugal force thereby applied to the liquid sample causes the liquid to flow along the slide surface and form a thin streak thereon.
  • the slide can be positioned at an angle from 10° to 70°, it is generally suitable for the slide to be positioned at an angle of 10° to 55°, and advisably 15° to 40°. It is advisable for the slide to maintain the same angle of inclination while spinning around the axis of rotation.
  • the speed of rotation about the axis is not narrowly critical. In the preparation of most slides about 500 to 5000 rpm, for a duration of about 0.1 to 3.0 seconds, is satisfactory, thereby creating a centrifugal force of between 500 and 2000 G. Optimal acceleration is 3000 rpm, for a duration of about 0.5 to 2.5 seconds. In general, as the slide angle increases from about 10° to 70° the speed of rotation can be decreased.
  • each slide usually substantially rectangular in shape, can be oriented so that its long parallel sides or shorter parallel sides if extended would intersect the axis of rotation. It is also advisable for the slide to be substantially tangential to the conical shell which is the path in which the slide travels during rotation. Square or rectangular cover slips can also be used in place of slides in the practice of the method.
  • the width of the thin streak which results from spinning the slide is approximately 0.5 mm to 3 mm, so that on an average microscope slide oriented with its long parallel sides intersecting the axis of rotation between one and six samples can be deposited on the surface of the slide to form separate thin streaks on one slide.
  • the slides are turned so that the shorter parallel sides intersect the axis, between ten and fifteen samples may be placed on one slide. This ability to allow such large numbers of individual liquid samples to be treated simultaneously diminishes the chances that variations in cell counts and subsequent diagnosis will be due to variances in technical procedure. Additionally, one or a plurality of slides can be centrifuged simultaneously in this method.
  • the method of the present invention may be practiced on any liquid desired to be microscopically viewed, including for example, biological fluids containing blood cells, bacterial cells, virus particles, and the like; chemical fluids; water and colloidal suspensions.
  • the liquid sample is a biological liquid containing cells
  • the volume of the sample suitable to provide a streak by the method can be 0.1 microliter to 5.0 microliters, with cell numbers as low as 10 2 cells per ml or as high as 10 9 cells per ml.
  • the liquid sample is other than biological, i.e., water, colloidal suspensions or chemicals
  • the volume suitable for practice of this method will vary with the viscosity and density of the liquid.
  • the thin streak which results from application of this method applied to cell-containing biological fluid is composed of cells confined to a narrow stripe or streak for part or all of the length of the slide, with most of the liquid having flowed to the end of the slide farthest from the axis of rotation, generally leaving a streak of single-cell thickness.
  • Excess liquid expelled from said slide by centrifugal force can be absorbed by placing any suitable means, such as an absorbent material near the edge of the slide towards which the sample flows.
  • a rotor for a centrifuge spinning apparatus for producing specimen slides for diagnostic evaluation with a slide support member on the rotor adapted to securely hold a slide at an angle of about 10° to 70° from the axis of rotation of the rotor with the face of the slide containing the liquid sample facing the axis of rotation, and with the radial distance from the sample to the axis of rotation being less than the greatest radial distance from one end of the slide to the axis of rotation so that spinning the rotor causes the slide to travel in a path which defines a truncated conical shell, with the slide substantially tangential to the shell so that the liquid sample can flow along the slide surface and form a thin streak thereon.
  • the rotor which can contain one or a plurality of said slide support members, can rotate about the axis of rotation in response to the operation of a motor capable of accelerating up to about 5000 rpm within about 0.1 second to 3.0 seconds and automatically shutting off.
  • Optimal acceleration is about 3000 rpm, within about 0.5 second to 2.5 seconds, followed by automatic shut-off.
  • the rotor will usually have one or more pairs of slide support members, it should be understood that the rotor can have one slide support member counterbalanced by suitable weight means, so that spinning of the rotor is achieved essentially free of vibration. Generally it is more convenient for the rotor to have paired opposing slide support members so that balancing can be more readily achieved. In this regard, even if only one slide is prepared, the rotor can be balanced by placing a dummy slide in the opposing slide support member.
  • the slide support members on the rotor can have side channels to prevent lateral displacement of the slide, and/or a flexible clip, or its equivalent, which extends over the top of said slide support members to prevent a slide from slipping out the top of said slide support member during centrifugation.
  • Each slide support member can also have an inwardly curved portion at the top for positioning means for absorbing excess liquid thrown off the slide during centrifugation.
  • One or more of the slide support members can include a mechanism for adjusting the slide angle within a range of 10° to 70° from said axis. The angular adjustment can be incremented, such as in 10° to 15° increments, or it can be infinitely variable from 10° to 70°.
  • each slide support member can have means for changing the orientation of a rectangular slide from a position in which the longer sides intersect the axis of rotation to a position in which the shorter parallel sides intersect that axis.
  • a secondary slide support member can be rotatable on said slide support member to change the slide orientation from a first position in which the long parallel sides of the rectangular slide define a line intersecting the axis of rotation to a second position wherein the slide is rotated 90° to allow that intersecting line to be defined by the short parallel sides of a rectangular slide.
  • the secondary slide support member can be attached to the slide support member by a bolt or threaded screw embedded in the secondary slide support member and extending through the back of the slide support member.
  • a securing device such as a butterfly nut, can be used to secure the secondary slide support member in the desired position by tightening the nut which is threaded on the screw to the back of the slide support member.
  • the butterfly nut can be turned to loosen the secondary slide support member.
  • the rotor 20 comprises two long, rectangular strips indicated generally at 22, 23 in Fig. 2, joined to rotatable shaft 38.
  • One strip 23 is superimposed on the other strip 22 at a 90° angle to define four identical outwardly extending radial slide support members 25, each oriented 90° from its adjacent slide support members.
  • the outer portions of said strips 22, 23 are bent at lateral lines 27 to define an angle X indicated generally at 34, from their axis of rotation, which is that of the rotatable shaft 38.
  • top edges 26 of the slide support members 25 are curved inwardly and operate in cooperation with the channels 24 and the intersection 31 of the slide support members 25 with the base 32 to retain the slide 28 within the slide support member 25 so that during centrifugal spinning, the centrifugal force exerted will not force the slide 28 out of the slide support member 25.
  • slide support members 25 are positioned at an angle X, 34, from the axis of rotation of rotatable shaft 38, which in Fig. 1 is a vertical axis of rotation although the axis can be horizontal or any angle between vertical and horizontal.
  • angle X is within the range of angular movement between 10° and 70° from the axis of rotation of the rotatable shaft 38.
  • Fig. 3 shows one orientation of a slide support member 25 and slide 28 when the slide 28 is positioned in the slide support member 25 in preparation for centrifugal spinning.
  • Slide 28 is oriented on the slide support member 25 with the surface of the slide 28 containing the liquid sample 30 facing the axis of rotation of rotatable shaft 38.
  • the radial distance from the sample 30, on the lower end of the slide 28, to the shaft 38 is less than the greatest radial distance 60 from the upper end of the slide 28 to the shaft 38, so that the spinning of the rotor 20 will cause the slide 28 to travel in a path which defines a truncated conical shell.
  • FIG. 4 an alternative embodiment of the slide support member 25 is shown.
  • a piece of absorbent filter paper 36 rests between the slide support member 25 and the slide 28.
  • the curved top 26 of the slide support member 25 functions to direct the pliable filter paper 36 in a tilted direction above the surface of slide 28 so that any excess liquid sample which is thrown off by centrifugal spinning will be captured and absorbed by the curved area of filter paper 36.
  • This is a particularly important function where said sample is an infectious sample, such as a bacterial culture or a blood or water sample containing infectious microorganisms.
  • infectious sample 30 is used to create a streak 42, that very little will be expelled from the slide 28.
  • the centrifugal force is applied in one direction to create a streak 42, no radial waste expulsion should occur.
  • FIG. 5 a cross-section of the slide support member 25 holding the slide 28, shows four spaced apart liquid samples 30 on the surface of the slide 28 which faces the rotatable shaft 38. Upon spinning the slide 28, streaks 42 are obtained on it as shown in Fig. 10.
  • Fig. 6 shows an alternate orientation of slide support members 25 on the rotor indicated generally at 20.
  • Slide support members 25 are shown to have an angle X from their axis of rotation, or rotatable shaft indicated at 38.
  • the angle X shown in the drawings is 15°.
  • the same principles of centrifugal force apply in this embodiment, since the centrifugal force acts on a sample 30 positioned on an inclined surface as seen in Fig. 7, which is a sectional view of slide support member 25.
  • Liquid sample 30 is positioned on the surface of slide 28 facing the rotatable shaft 38, but at the upper part of the slide 28.
  • the liquid sample 30 is still at a position where the radial distance from the sample 30 to the shaft 38 is less than the greatest radial distance 60 from one end of the slide 28 to the shaft 38.
  • spinning of the rotor 20 causes the slide 28 to travel in a path which defines a truncated conical shell, so that the liquid sample 30 can flow along the slide surface and form a thin streak 42 thereon.
  • Fig. 8 illustrates another embodiment of slide support member 25.
  • This Figure which is an enlarged section of the top part of a slide support member 25, there is fastened onto the back of the slide support member 25 a flexible clip 44.
  • the flexible clip 44 functions as additional retention means to prevent slide 28 from flying from out the top of slide support member 25 during centrifugal spinning.
  • the flexible clip 44 is preferably formed of thin, flexible steel, so that it is able to be pressed towards the slide support member 25 out of the path of insertion and removal of the slide 28 into the slide support member 25.
  • the flexible clip 44 is seen from the back view, where the rivets 46, which hold the clip 44 onto the back of the slide support member 25, may clearly be seen.
  • Fig. 10 shows the end result of the use of the method and apparatus of the present invention.
  • a plurality of sample specimens 30 are dotted on the slide 28, as shown in Fig. 5, and then subjected to centrifugal spinning at an angle X, 34, from the axis of rotation of rotatable shaft 38.
  • the resulting centrifugal force acting on the specimen sample 30 lying on an inclined plane spreads the sample 30 in a thin streak 42 along the length of the slide 28.
  • Fig. 10 shows four such streaks 42, resulting from subjecting four liquid samples 30 as in Fig. 5 on one slide 28 to the method of the present invention, thereby allowing exposure of the samples to uniform testing conditions.
  • Fig. 11 shows an alternative embodiment of a slide support member 25 on a rotor indicated generally at 20.
  • the slide support member 25 is adapted for adjustable angular movement by means of a leg 52 attached to the back of the slide support member 25 by means of a hinge 48.
  • the slide support member 25 is attached for adjustable movement to the base 32 of the rotor 20.
  • the base 32 extends beyond the slide support member 25 in this embodiment.
  • Positioned at stated points along the base 32 are slots 54, 56, and 58, for receiving the foot 62 of the adjustable leg 52.
  • angle X defines an angle of 15° from the axis of rotation of rotatable shaft 38.
  • angle X of slide support member 25 from the shaft 38 is 25°.
  • angle X reaches 35° from the shaft 38.
  • Figs. 12 shows another embodiment of the rotor of the present invention in which slide support members 25 also provide means for changing the orientation of a rectangular slide to encompass a greater number of specimens.
  • Attached to said slide support members 25 are secondary slide support members 80 which are rotatable from a first position indicated generally at 92 in Figs. 12 and 14 in which a rectangular slide is held so that its longer parallel sides define a line which intersects the axis of rotation represented by rotatable shaft 38 to a second position indicated generally at 90 in Fig. 13 and in dotted lines in Fig. 12 in which said secondary slide support member 80 holds a rectangular slide so that the short parallel sides of the rectangular slide define the line which intersects the axis of rotation 38.
  • Said secondary slide support member 80 is joined to the slide support member 25 by a bolt 82 which extends from the secondary slide support member 80 through the back of the slide support member 25.
  • Said bolt 82 is releasably tightened by butterfly nut 84 to hold said secondary slide support member 80 in said first or said second positions, 92 and 90 respectively.
  • said secondary slide support member 80 has channels 96 on opposing edges of said secondary slide support member 80 spaced apart to loosely receive the opposing longitudinal edges of a conventional rectangular microscope slide 28.
  • the secondary slide support member 80 can be rotated from its first position 92 illustrated in Fig. 14 by loosening butterfly nut 84, turning said secondary slide support member 80 to the second position 90, illustrated in Fig. 13, and turning butterfly nut 84 tightly against slide support member 25 to maintain the position of secondary slide support member 80 during centrifugation.
  • Fig. 15 shows the end result of the use of the method and apparatus of the present invention in the embodiment of Fig. 12 having means to change the orientation of a rectangular slide 28.
  • a plurality of sample specimens are dotted on slide 28 and then subjected to centrifugal spinning and an angle X, 34, from the axis of rotation of rotatable shaft 38, the secondary slide support member 80 being in the second position 90 illustrated in Fig. 13.
  • Resulting centrifugal force acts on the specimen samples lying on an inclined plane to spread the samples in thin streaks 42 along the surface of the slide 28.
  • Fig. 15 shows fifteen such streaks 42 resulting from subjecting fifteen liquid samples on one slide 28 in the second secondary slide support member position 90 in which the shorter parallel sides of slide 28 when in the secondary slide support member 80 define a line which would intersect the axis of rotation.
  • the following example illustrates use of the method and apparatus of the present invention with a biological cell-containing liquid, blood.
  • Fig. 1 of the drawing two slides are prepared for microscopic examination of a blood sample.
  • Four drops of blood sample containing approximately 10 4 cells per cubic millimeter are deposited near the bottom end of slide A, the drops being equally spaced from each other across the width of the slide.
  • slide B The same procedure is followed for slide B.
  • Each slide is slipped into one opposing slide support member of the rotor beneath the grip of the channels which line the longitudinal length of the slide support members.
  • Filter paper sections are placed on the curved tips of the slide support members which curve the filter paper slightly over the top of each slide.
  • the slides are positioned in the opposing slide support members so that the drops of blood are closer to the base of the rotor.
  • the slide support members in this rotor are angled 15° from the axis of rotation.
  • the rotor is powered by a conventional motor and set to achieve 2000 rpm for 1.5 seconds, creating a force of gravity of about 700 G.
  • the slides, stained with appropriate dyes and cytochemical reagents, are inserted onto the base of a microscope and examined at a magnitude of 400, in which three microscope fields are seen at once.
  • the laboratory technician is able to follow the length of the streak, since under this magnification the entire width of each streak can be observed.
  • Observable in each streak are the individual blood cells which can be differentiated by their characteristic morphology. Since the cells are spread in a single layer and the number of cells per oil immersion field is high (10-15 cells per field in contrast to 1-2 cells per field by wedge technique), it is quite easy to count the number of cells per sample drop by moving the platform of the microscope in one direction to follow the length of each streak formed. Both slides are examined by this method and differential counts are made quickly and easily.

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  • Physics & Mathematics (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Sampling And Sample Adjustment (AREA)
  • Centrifugal Separators (AREA)
  • Microscoopes, Condenser (AREA)
EP83108028A 1982-08-18 1983-08-13 Procédé et dispositif de préparation d'une lamelle de microscope portant un échantillon Withdrawn EP0103190A3 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US06/409,157 US4468410A (en) 1982-08-18 1982-08-18 Method and apparatus for producing a microscopic specimen slide
US409157 1982-08-18

Publications (2)

Publication Number Publication Date
EP0103190A2 true EP0103190A2 (fr) 1984-03-21
EP0103190A3 EP0103190A3 (fr) 1984-07-18

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EP83108028A Withdrawn EP0103190A3 (fr) 1982-08-18 1983-08-13 Procédé et dispositif de préparation d'une lamelle de microscope portant un échantillon

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US (1) US4468410A (fr)
EP (1) EP0103190A3 (fr)
JP (1) JPS59126227A (fr)

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CN113029719A (zh) * 2021-03-10 2021-06-25 山东大学 一种岩土模型试验件用制备装置及方法

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113029719A (zh) * 2021-03-10 2021-06-25 山东大学 一种岩土模型试验件用制备装置及方法
CN113029719B (zh) * 2021-03-10 2022-03-25 山东大学 一种岩土模型试验件用制备装置及方法

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EP0103190A3 (fr) 1984-07-18
JPS59126227A (ja) 1984-07-20
US4468410A (en) 1984-08-28

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